Combination therapies for the treatment of alzheimer&#39;s disease and related disorders

ABSTRACT

The present invention relates to combination therapies for treating Alzheimer&#39;s disease or an amyloidosis-associated pathological condition comprising co-administering a therapeutically effective amount of a first compound, and a therapeutically effective amount of a second compound. In certain embodiments, the first compound or the second compound inhibits AB peptide polymerization; is an anti-inflammatory; improves cognitive function, mood, or social behavior; is associated with Tau or alpha-synuclein; or regulates amyloid peptide washout.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/902,486, filed Feb. 22, 2018, now U.S. Pat. No.10,413,551, issued Sep. 17, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/046,486, filed Feb. 18, 2016, now U.S. Pat. No.9,913,847, issued Mar. 13, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/437,316, now U.S. Pat. No. 9,855,276, issuedJan. 2, 2018, which represents the national stage entry of PCTInternational Application No. PCT/US2013/66069, filed Oct. 22, 2013,which claims the benefit of U.S. provisional Application No. 61/718,303,filed Oct. 25, 2012, the contents of each of which are expresslyincorporated by reference in their entirety.

BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disorder ofthe brain, which is characterized by the memory deterioration,behavioral disturbances, impairment of activities of daily living, andloss of independent function. It is thought that 18-24 million people inthe world are currently suffering from AD, two-thirds of whom are livingin developed or developing countries. This number is expected to reach34 million by 2025.

AD is a complicated disease. It may even be the result of more than onedisease. It is characterized by an accumulation otinsoluble aggregatesof amyloid-beta peptide (Aβ). such as Aβ oligomers. These aggregates oroligomers are associated with cell inflammatory response and are thoughtto bind to a surface receptor on neurons and change the structure of thesynapse, thereby disrupting neuronal communication. Due to the minuteamount produced per day (22-27 ng/day) and accumulated for years (about7-10 mg in brains of AD subjects), this daily inflammatory response isinvisible and not associated with any major symptoms. In addition, tauprotein abnormalities are thought to play a role in the disease cascade.Hyperphosphorylated tau proteins are thought to pair with other threadsof tau. Eventually, they form neurofibrillary tangles inside nerve cellbodies. When this occurs, the microtubules disintegrate, collapsing theneuron's transport system. This may result first in malfunctions inbiochemical communication between neurons and later in the death of thecells.

The recent failures of several promising drugs have spurred greaterurgency to investigate new targets and their interconnectedness. Thatsaid, new therapies for Alzheimer's are needed.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a method of treating adisease or condition in a subject in need thereof comprisingco-administering a therapeutically effective amount of a first compound,and a therapeutically effective amount of a second compound, wherein thedisease or condition is Alzheimer's disease, dementia, anamyloidosis-associated condition, or a head injury.

In certain embodiments, the invention relates to a method of slowing theprogression of a disease or condition in a subject in need thereofcomprising co-administering a therapeutically effective amount of afirst compound, and a therapeutically effective amount of a secondcompound, wherein the disease or condition is Alzheimer's disease,dementia, an amyloidosis-associated condition, or a head injury.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound inhibits Aβ peptidepolymerization; and the second compound is an anti-inflammatory.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound is ananti-inflammatory; and the second compound improves cognitive function,mood, or social behavior.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound inhibits Aβ peptidepolymerization; and the second compound improves cognitive function,mood, or social behavior.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound is ananti-inflammatory; and the second compound is associated with Tau oralpha-Synuclein.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound is ananti-inflammatory; and the second compound modulates amyloid peptideformation and washout.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound and the secondcompound inhibit Aβ peptide polymerization.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound and the secondcompound are anti-inflammatories.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound and the secondcompound improve cognitive function, mood, or social behavior.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound and the secondcompound are associated with Tau or alpha- Synuclein.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first compound and the secondcompound modulate amyloid peptide formation and washout.

In certain embodiments, the compound inhibiting Aβ peptidepolymerization is selected from the group consisting of formula I-IV:

wherein, independently for each occurrence,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹° are hydrogen, halo, azido,alkyl, haloalkyl, perhaloalkyl, fluoroalkyl, perfluoroalkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl,heteroaralkyl, hydroxy, alkoxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, amino, alkylamino, arylamino, acylamino,heteroarylamino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, acyl, carboxyl, oxycarbonyl, acyloxy, silyl, thioether,sulfo, sulfonate, sulfonyl, sulfonamido, formyl, cyano, isocyano, or-Y-(haloalkylene)-alkyl; R⁷ is hydrogen, halo, azido, alkyl, haloalkyl,perhaloalkyl, fluoroalkyl, perfluoroalkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl,hydroxy, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy,amino, alkylamine, arylamino, acylamino, heteroarylamino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, acyl, carboxyl,oxycarbonyl, acyloxy, silyl, thioether, sulfo, sulfonate, sulfonyl,sulfonamide, formyl, cyano, isocyano, -Y-(haloalkylene)-alkyl, or-Y-(haloalkylene)-R; R^(N) is hydrogen, lower alkyl, or-(haloalkylene)-alkyl; Y is a bond, N(R^(N)), O, or S; and

R is

provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, or R¹⁰is -Y-(haloalkylene)-alkyl; or R^(N) is -(haloalkylene)-alkyl.

In certain embodiments, the Aβ peptide polymerization inhibitor is

In certain embodiments, the Aβ peptide polymerization inhibitor is

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts various functions of mast cell agents.

FIG. 2 depicts a three-drug combination.

FIG. 3 depicts various therapeutic agents of the invention and theirrespective proposed mechanisms of action in monotherapy.

FIG. 4 depicts the water maze recorded data of in vivo cromolyn andibuprofen treatment of transgenic mice-modeling like Alzheimer'sDisease. The results indicate that treated transgenic mice have closelybehavior to wild type normal control group.

FIG. 5A illustrates the measurement of TBS soluble Aβ level by WAKOELISA. The experiments show that TBS Aβ level decreases following bytreatment of cromolyn sodium with dose-dependency and shows that Aβ-40level decreases following by treatment of cromolyn sodium withdose-dependency.

FIG. 5B illustrates the measurement of TBS soluble Aβ level by WAKOELISA. The experiments show that TBS Aβ level decreases following bytreatment of cromolyn sodium with dose-dependency and shows that Aβ-42level decreases following by treatment of cromolyn sodium withdose-dependency. N=3 or 5 animals per group, average ±SE. The p value issignificant using one-way ANOVA test (Bonferroni's test). Both of totalsoluble Aβ (as shown as Gdn+) and monomeric Aβ (as shown as Gdn−)decease after the addition of cromolyn sodium. The dose of 2.1 mg/kg ofcromolyn sodium was enough to decrease TBS soluble Aβ.

FIG. 6A illustrates the measurement of TBS soluble Aβ oligomer level IBLoligomer ELISA. The experiments show that Aβ oligomer level was notchanged following the treatment with cromolyn sodium and shows theexperiments of IBL Aβ oligomer ELISA (82E1-82E1).

FIG. 6B illustrates the measurement of TBS soluble Aβ oligomer level IBLoligomer ELISA. The experiments show that Aβ oligomer level was notchanged following the treatment with cromolyn sodium and show thedifference the experiments with Gdn and those without Gdn using Aβ WAKOELISA. N=3 or 5 animals per group, average±SE. The p value is notsignificant using one-way ANOVA test (Bonferroni's test). Both ELISA(IBL oligomer ELISA and the differences between with and without Gdnusing WAKO ELISA) showed that oligomer level was not changed followingthe treatment with cromolyn sodium.

FIG. 6C illustrates the measurement of TBS soluble Aβ oligomer level IBLoligomer ELISA. The experiments show that Aβ oligomer level was notchanged following the treatment with cromolyn sodium and show thedifference the experiments with Gdn and those without Gdn using Aβ WAKOELISA. N=3 or 5 animals per group, average±SE. The p value is notsignificant using one-way ANOVA test (Bonferroni's test). Both ELISA(IBL oligomer ELISA and the differences between with and without Gdnusing WAKO ELISA) showed that oligomer level was not changed followingthe treatment with cromolyn sodium.

FIG. 7 illustrates the biodistribution of cromolyn Compound A followingintravenous injection in mice. In FIG. 7, a 5, 30 or 60 minute,corresponding to Series 1, 2 or 3, respectively in the graph, brainuptake shows 1% accumulation with little or no washout for the periodmeasured.

FIG. 8 illustrates Aβ aggregation test in the absence of cromolyn. Theexperiment was assayed by thioflavin fluorescent intensity kinetics.

FIG. 9 illustrate Aβ aggregation test after the addition of cromolyn(CO399) or its ¹⁹F derivative (TS734). The addition of cromolyn (CO399)and its ¹⁹F derivative (TS734) at nanomolar concentration showsinhibition of Aβ aggregation.

FIG. 10 illustrates the side view of the relative structures andlocations of cromolyn and Aβ after cromolyn binds Aβ through a bindingmodel simulation.

FIG. 11 illustrate the top view of the relative structures and locationsof cromolyn and Aβ after cromolyn binds Aβ through a binding modelsimulation.

DESCRIPTION OF THE INVENTION Therapeutic Agents

Featured herein are methods of treating or preventing anamyloidosis-associated condition in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of atleast two compounds selected from the group consisting of an Aβ peptidepolymerization inhibitor, an anti-inflammatory; a compound that improvescognitive function, mood, or social behavior, a compound associated withTau or alpha-Synuclein and a compound that regulates amyloid peptidewashout. The methods involve multifunctional treatment combinations anddosing.

These combination treatments may slow down memory loss or braindegeneration in early stages of AD. For example, a subject may beexhibiting mild cognitive impairment (MCI), or may have a narrow MMSE(mini-mental state examination) score of between about 24 and about 28.

These combination treatments may also be administered to subjects withAD. In certain embodiments, the subjects experience improved quality oflife.

Aβ—Peptide Polymerization Inhibitors

Thioflavin or[N-methyl-(¹¹C)]2-(4′-methylaminophenyl)-6-hydroxybenzothiazole(PIB) isan Aβ peptide polymerization inhibitor.

Aβ peptide polymerization inhibitors are also represented by formulaI-IV:

wherein, independently for each occurrence,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are hydrogen, halo, azido,alkyl, haloalkyl, perhaloalkyl, fluoroalkyl, perfluoroalkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl,heteroaralkyl, hydroxy, alkoxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, amino, alkylamino, arylamino, acylamino,heteroarylamino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, acyl, carboxyl, oxycarbonyl, acyloxy, silyl, thioether,sulfo, sulfonate, sulfonyl, sulfonamido, formyl, cyano, isocyano, or-Y-(haloalkylene)-alkyl;

R⁷ is hydrogen, halo, azido, alkyl, haloalkyl, perhaloalkyl,fluoroalkyl, perfluoroalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxy, alkoxy,aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, amino, alkylamine,arylamino, acylamino, heteroarylamino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, acyl, carboxyl, oxycarbonyl, acyloxy, silyl,thioether, sulfo, sulfonate, sulfonyl, sulfonamide, formyl, cyano,isocyano, -Y-(haloalkylene)-alkyl, or -Y-(haloalkylene)-R;

R^(N) is hydrogen, lower alkyl, or -(haloalkylene)-alkyl;

Y is a bond, N(R^(N)), O, or S; and

R is

provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, or R¹°is -Y-(haloalkylene)-alkyl; or R^(N) is -(haloalkylene)-alkyl.

In certain embodiments, the Aβ peptide polymerization inhibitor is

In certain embodiments, the Aβ peptide polymerization inhibitor is

The following U.S. patents and patent applications, which are herebyincorporated by reference in their entirety, also describe Aβ peptidepolymerization inhibitors: U.S. Pat. Nos. 7,858,803; 6,972,127;6,946,116; 6,696,039; 6,168,776; 5,594,142; 4,481,206; 4,405,735; andU.S. Patent Application Publication No. 2011/0060138.

Additional Aβ peptide polymerization inhibitors may be selected from thegroup consisting of:

A gamma secretase inhibitor, such as LY451039 (Semagacestat, Eli Lily)may also function as an Aβ peptide polymerization inhibitor. Metalionophores, such as PBT2 (Prana), which target metal-induced aggregationof Aβ may also function as an Aβ peptide polymerization inhibitor.Statins may also function as Aβ peptide polymerization inhibitors.

Endocannabinoids, such as arachidonoylethanolamine,tetrahydrocannabinol, 2-arachidonoyl glycerol, 2-arachidonyl glycerylether, N-arachidonoyl-dopamine, or virodhamine are further examples ofAβ peptide polymerization inhibitors.

An appropriate Aβ peptide polymerization inhibitor should slow the rateof Aβ peptide polymerization by at least about three times slower, aboutfive times slower, about seven times slower, about 10 times slower,about 15 times slower, about 20 times slower, about 25 times slower,about 30 times slower, about 35 times slower, about 40 times slower,about 45 times slower, about 50 times or about 100 times slower than therate of A13 peptide polymerization in the absence of the inhibitor.

An appropriate Aβ peptide polymerization inhibitor should haveappropriate structures (size, lipophilicity, and charge) to allow forpenetration of the blood brain barrier (BBB). In addition the Aβ peptidepolymerization inhibitor may have specific affinity for solubilizing andinteracting with oligomers to prevent them from aggregating.

The daily required dose administration should be proportional to theapproximate daily quantity of Aβ peptide; this dosing regimen minimizesside effects from extensive dosing. Appropriate logP, polar surface area(PSA) and % PSA should determine brain permeability and drugeffectiveness.

An estimation of the amount of deposited amyloid-β in the brain requiresdata that have a sufficient sample size and are derived fromquantitative assay systems that are combined with aggressive, formicacid extraction protocols. Assuming that the average weight of an ADbrain is 1,150 g and the grey matter of the cortex, which contains themajority of deposited amyloid-β, comprises 42% of the weight of thebrain, Gravina et al. calculated that -10 mg of amyloid-β per brain isdeposited, whereas Naslund et al. calculated that ˜4 mg of amyloid-β perbrain is deposited. Although the result obtained by Naslund et al. islower than other literature estimates, for the purposes of this analysisthe total amount of Aβ in a human AD brain at end-stage disease isassumed to be derived from 10 mg of Aβ-plaque.

It is important to compare the amount of amyloid-β that is deposited inthe AD brain with the overall rate of amyloid-β production, to provide aconceptual framework for this aspect of the disease process and to placeinto context the potential for different amyloid-β-centric therapeuticsto mediate a therapeutic effect. Therefor the estimated amount producedper day is less than 25 ng per day.

In certain embodiments, the Aβ peptide polymerization inhibitor isadministered in nM concentrations.

Anti-Inflammatory Compounds

Anti-inflammatory compounds may be mast cell stabilizers, such ascromolyn, a cromolyn derivative, a cromolyn analog, such as thosedescribed in U.S. Patent Application Publication No. 2012/0058049, whichis hereby incorporated by reference in its entirety, eugenol,nedocromil, pemirolast, olopataidne, alfatoxin G₁ alfatoxin B1,alfatoxin Mi deoxynivalenol, zearalenone, ochratoxin A, fumonisin B₁hydrolyzed fumonisin B₁ patulin, ergotamine,

Anti-inflammatory compounds may also be a non-steroidalanti-inflammatory drug (NSAID), such as acetylsalicylic acid,diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen,ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen,indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac,nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam,isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, celecoxib, licofelone, hyperforin, or figwort.

Since the inflammatory response to Aβ peptide production has invisibleside effect symptoms, dosing control is important for preventingsystemic side effect toxicity and worsening outcome.

Compounds That Improve Cognitive Function, Mood and/or Social Behavior

Agents for improving cognitive function, mood, and/or social behaviorinclude cholinesterase inhibitors, such as donepezil, riastigmine, orgalantamine. Other examples include N-methyl-D-aspartate (NMDA) receptorantagonists, such as memantine. Antioxidants, such as vitamin E orselegiline may also improve a subject's cognitive function, mood and/orsocial behavior. Allopregnanolone, a neurosteroid present in the bloodis another example of an agent that improves cognitive function, mood,or social behavior in a subject.

Agents that initiate and/or amplify a subject's immune response, such asa tumor necrosis factor (TNF) inhibitor, e.g. etanercept or antibiotics,such as doxycycline, rifampin, or minocycline, are also agents thatimprove cognitive function, mood, or social behavior.

Spiro-(N′-methyl-piperidyl-4′)-N-ethyl-succinimide, as described in U.S.Pat. No. 4,481,206, which is hereby incorporated by reference in itsentirety, is another example of an agent that improves cognitivefunction, mood or social behavior. This molecule improves cognitivefunction by compensatory activation of other receptors for improvingnerve communication and cognition. These agents are traditionally usedto improve the quality of life of subjects with onset and diseaseprogression.

Latrepirdine appears to operate through multiple mechanisms of action,both blocking the action of neurotoxic beta-amyloid proteins andinhibiting L-type calcium channels, modulating the action of AMPA andNMDA glutamate receptors, and may exert a neuroprotective effect byblocking a novel target that involves mitochondrial pores, which arebelieved to play a role in the cell death that is associated withneurodegenerative diseases and the aging process.

R3487 (Roche) is a partial agonist of the nicotinic alpha-7 receptor, ahighly specialized receptor found in the central nervous system. In arecently completed Phase 2a study in Alzheimer's disease patients, R3487demonstrated a statistically significant effect on multiple measures ofcognition.

Agents Associated with Tau or Alpha-Synuclein

Methylthioninium chloride, an inhibitor of tau protein aggregation is anexample of an agent that is associated with tau or alpha-synuclein.Agents that may stabilize tau while part of the tubular nerve system mayslow down the production of intra neuron fibrilary tangles and slow downthe progression of the disease.

Agents that Regulate Amyloid Peptide Washout

AP-peptide specific antibodies, although they cannot penetrate thenormal BBB, can cause equilibrium changes between the amount of Aβoligomers in the brain, cerebrospinal fluid (CSF), and vascular system.The antibodies can bind and remove the Aβ peptide in CSF and blood andcause the equilibrium to favor washout of the Aβ peptide from brain.While many of these antibodies display significant toxicity and sideeffects, the toxicity is largely due to the large doses required.

Examples of Aβ-peptide specific antibodies include: bapineuzumab(Elan/Johnson & Johnson), solanezumab (LY2062430) (Eli Lilly),gammaglobulin IV (Baxter), and PF-4360365 (Pfizer).

ACC-001 (Elan/Johnson & Johnson) is an anti-beta amyloid vaccine; itstimulates the immune system to attack beta-amyloid.

Similarly, siRNA that targets Aβ peptide may be used. Some AD conditionsand brain injury cause a breakdown in the BBB; therefore siRNA canpenetrate the brain and silence AP peptide production.

CERE-110 (Ceregene Inc.) is nerve growth factor (NGF) gene therapy. NGFspecifically targets basal forebrain cholinergic neurons, which releaseacetylcholine (Ach) in the cerebral cortex and hippocampus. Preclinicaldata in rats demonstrate that NGF prevented cholinergic neuron celldeath and reversed age-related behavioral decline. NGF gene therapy hasbeen tested in rhesus monkeys, and these studies demonstrated that NGFameliorates cholinergic neuron atrophy and restores cholinergic axonaldensity in aged monkeys to levels observed in young monkeys.

Semagacestat (LY450139) is a gamma-secretase inhibitor; gamma secretaseis responsible for proteolysis of amyloid precursor protein (APP).Proteolysis of APP forms Aβ.

Another gamma secretase inhibitor is NIC5-15 (Humanetics).

Therapeutic Methods

Combinations of the compounds described above may be administered to asubject in a single dosage form or by separate administration of eachactive agent. The agents may be formulated into a single tablet, pill,capsule, or solution for parenteral administration and the like.Individual therapeutic agents may be isolated from other therapeuticagent(s) in a single dosage form. Formulating the dosage forms in such away may assist in maintaining the structural integrity of potentiallyreactive therapeutic agents until they are administered. Therapeuticagents may be contained in segregated regions or distinct caplets or thelike housed within a capsule. Therapeutic agents may also be provided inisolated layers in a tablet.

Alternatively, the therapeutic agents may be administered as separatecompositions, e.g., as separate tablets or solutions. One or more activeagent may be administered at the same time as the other active agent(s)or the active agents may be administered intermittently. The length oftime between administrations of the therapeutic agents may be adjustedto achieve the desired therapeutic effect. In certain instances, one ormore therapeutic agent(s) may be administered only a few minutes (e.g.,about 1, 2, 5, 10, 30, or 60 min) after administration of the othertherapeutic agent(s). Alternatively, one or more therapeutic agent(s)may be administered several hours (e.g., about 2, 4, 6, 10, 12, 24, or36 h) after administration of the other therapeutic agent(s). In certainembodiments, it may be advantageous to administer more than one dosageof one or more therapeutic agent(s) between administrations of theremaining therapeutic agent(s). For example, one therapeutic agent maybe administered at 2 hours and then again at 10 hours followingadministration of the other therapeutic agent(s). The therapeuticeffects of each active ingredient should overlap for at least a portionof the duration, so that the overall therapeutic effect of thecombination therapy is attributable in part to the combined orsynergistic effects of the combination therapy.

The dosage of the active agents will generally be dependent upon anumber of factors including pharmacodynamic characteristics of eachagent of the combination, mode and route of administration of activeagent(s), the health of the patient being treated, the extent oftreatment desired, the nature and kind of concurrent therapy, if any,and the frequency of treatment and the nature of the effect desired. Ingeneral, dosage ranges of the active agents often range from about 0.001to about 250 mg/kg body weight per day. For a normal adult having a bodyweight of about 70 kg, a dosage may range from about 0.1 to about 25mg/kg body weight. However, some variability in this general dosagerange may be required depending upon the age and weight of the subjectbeing treated, the intended route of administration, the particularagent being administered and the like. Since two or more differentactive agents are being used together in a combination therapy, thepotency of each agent and the interactive effects achieved using themtogether must be considered. Importantly, the determination of dosageranges and optimal dosages for a particular mammal is also well withinthe ability of one of ordinary skill in the art having the benefit ofthe instant disclosure.

Dosage ranges for agents may be as low as 5 ng/d. In certainembodiments, about 10 ng/day, about 15 ng/day, about 20 ng/day, about 25ng/day, about 30 ng/day, about 35 ng/day, about 40 ng/day, about 45ng/day, about 50 ng/day, about 60 ng/day, about 70 ng/d, about 80ng/day, about 90 ng/day, about 100 ng/day, about 200 ng/day, about 300ng/day, about 400 ng/day, about 500 ng/day, about 600 ng/day, about 700ng/day, about 800 ng/day, about 900 ng/day, about 1 μg/day, about 2μg/day, about 3 μg/day, about 4 μg/day, about 5 μg/day, about 10 μg/day,about 15 μg/day, about 20 μg/day, about 30 μg/day, about 40 μg/day,about 50 μg/day, about 60 μg/day, about 70 μg/day, about 80 μg/day,about 90 μg/day, about 100 μg/day, about 200 μg/day, about 300 μg/day,about 400 μg/day, about 500 μg/day, about 600 μg/day, about 700 μg/day,about 800 μg/day, about 900 μg/day, about 1 mg/day, about 2 mg/day,about 3 mg/day, about 4 mg/day, about 5 mg/day, about 10 mg/day, about15 mg/day, about 20 mg/day, about 30 mg/day, about 40 mg/day, or about50 mg/day of an agent of the invention is administered.

In certain embodiments, the agents of the invention are administered inpM or nM concentrations. In certain embodiments, the agents areadministered in about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM,about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 300 pM,about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM,about 900 pM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM,about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM,about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, orabout 900 nM concentrations.

In certain embodiments, the size of the active agent is important. Incertain embodiments, the active agent is less than about 3 μm, less thanabout 2 μm, less than about 1 μm in diameter. In certain embodiments,the active agent is from about 0.1 μm to about 3.0 μm in diameter. Incertain embodiments, the active agent is from about 0.5 μm to about 1.5μm in diameter. In certain embodiments, the active agent is about 0.2μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7μm, about 0.8 μm, about 0.9 μm, about 1.0 μm, about 1.1 μm, about 1.2μm, about 1.3 μm, about 1.4 μm, or about 1.5 μm in diameter.

It may be advantageous for the pharmaceutical combination to becomprised of a relatively large amount of the first component comparedto the second component. In certain instances, the ratio of the firstactive agent to second active agent is about 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 90:1, 80:1, 70:1, 60:1,50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. Itfurther may be preferable to have a more equal distribution ofpharmaceutical agents. In certain instances, the ratio of the firstactive agent to the second active agent is about 4:1, 3:1, 2:1, 1:1,1:2, 1:3, or 1:4. It also may be advantageous for the pharmaceuticalcombination to have a relatively large amount of the second componentcompared to the first component. In certain instances, the ratio of thesecond active agent to the first active agent is about 30:1, 20:1, 15:1,10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. In certain instances, the ratio of thesecond active agent to first active agent is about 100:1, 90:1, 80:1,70:1, 60:1, 50:1, or 40:1. In certain instances, the ratio of the secondactive agent to first active agent is about 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, or 110:1. A composition comprisingany of the above-identified combinations of first therapeutic agent andsecond therapeutic agent may be administered in divided doses about 1,2, 3, 4, 5, 6, or more times per day or in a form that will provide arate of release effective to attain the desired results. The dosage formmay contain both the first and second active agents. The dosage form maybe administered one time per day if it contains both the first andsecond active agents.

For example, a formulation intended for oral administration to humansmay contain from about 0.1 mg to about 5 g of the first therapeuticagent and about 0.1 mg to about 5 g of the second therapeutic agent,both of which are compounded with an appropriate and convenient amountof carrier material varying from about 5 to about 95 percent of thetotal composition. Unit dosages will generally contain between about 0.5mg to about 1500 mg of the first therapeutic agent and 0.5 mg to about1500 mg of the second therapeutic agent. The dosage may be about 25 mg,50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000mg, etc., up to about 1500 mg of the first therapeutic agent. The dosagemay be about 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600mg, 800 mg, or 1 000 mg, etc., up to about 1500 mg of the secondtherapeutic agent.

Definitions

As used herein, the following terms and phrases should have the meaningsprovided below.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

An “amyloidosis-associated condition” is a disease that is associatedwith amyloid deposition and can include but not be limited toAlzheimer's Disease, idiopathetic myeloma, amyloid polyneuropathy,amyloid cardiomyopathy, systemic senile amyloidosis, amyloidpolyneuropathy, hereditary cerebral hemorrhage with amyloidosis, Down'ssyndrome, Scrapie, medullary carcinoma of the thyroid, isolated atrialamyloid, β₂-microglobulin amyloid in dialysis patients, inclusion bodymyositis, β₂-amyloid deposits in muscle wasting disease, and Islets ofLangerhans diabetes Type I1 insulinoma. Type 2 diabetes mellitus,hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A(reactive), secondary amyloidosis, familial Mediterranean fever,familial amyloid nephropathy with urticaria and deafness (Muckle-wellsSyndrome), amyloid lambda L-chain or amyloid kappa L-chain (idiopathic,myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, systemic senile amyloidoses, AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-1 (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice), headinjuries (traumatic brain injury), dementia, fibrinogen- associatedamyloid; and Asor or Pr P-27 (scrapie, Creutzfeld Jacob disease,Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis)or in cases of persons who are homozygous for the apolipoprotein E4allele, and the condition associated with homozygosity for theapolipoprotein E4 allele or Huntington's disease.

“Amyloidosis” is a condition characterized by the accumulation ofvarious insoluble, fibrillar proteins in the tissues of a patient. Anamyloid deposit is formed by the aggregation of amyloid proteins,followed by the further combination of aggregates and/or amyloidproteins.

Many forms of amyloidosis exist, and the disease can be classified intofour groups: primary amyloidosis, secondary amyloidosis, hereditaryamyloidosis, and amyloidosis associated with normal aging. Primaryamyloidosis (light chain amyloidosis) occurs with abnormalities ofplasma cells, and some people with primary amyloidosis also havemultiple myeloma (cancer of the plasma cells). Typical sites of amyloidbuildup in primary amyloidosis are the heart, lungs, skin, tongue,thyroid gland, intestines, liver, kidneys, and blood vessels. Secondaryamyloidosis may develop in response to various diseases that causepersistent infection or inflammation, such as tuberculosis, rheumatoidarthritis, and familial Mediterranean fever. Typical sites of amyloidbuildup in secondary amyloidosis are the spleen, liver, kidneys, adrenalglands, and lymph nodes. Hereditary amyloidosis has been noted in somefamilies, particularly those from Portugal, Sweden, and Japan. Theamyloid-producing defect occurs because of mutations in specificproteins in the blood. Typical sites for amyloid buildup in hereditaryamyloidosis are the nerves, heart, blood vessels, and kidneys.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

The terms “augmentation” or “augment” refer to combinations where one ofthe compounds increases or enhances therapeutic effects of anothercompound or compounds administered to a patient. In some instances,augmentation can result in improving the efficacy, tolerability, orsafety, or any combination thereof, of a particular therapy.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The definition of each expression, e.g., alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

A comprehensive list of the abbreviations utilized by organic chemistsof ordinary skill in the art appears in the first issue of each volumeof the Journal of Organic Chemistry; this list is typically presented ina table entitled Standard List of Abbreviations.

The terms “hydroxy” and “hydroxyl” refer to the group —OH.

The term “oxo” refers to the group ═O.

The term “carboxylate” or “carboxyl” refers to the group —COO⁻ or —COOH.

The term “cyano” refers to the group —CN.

The term “nitro” refers to the group —NO₂.

The term “amino” refers to the group —NH₂.

The term “acyl” or “aldehyde” refers to the group —C(═O)H.

The term “amido” or “amide” refers to the group —C(O)NH₂.

The term “aminoacyl” or “acylamino” refers to the group —NHC(O)H.

The term “thiol” refers to the group —SH.

The term “thioxo” refers to the group ═S.

The term “sulfinyl” refers to the group —S(═O)H.

The term “sulfonyl” refers to the group —SO₂H.

The term “sulfonylamido” or “sulfonamide” refers to the group —SO₂NH₂.

The term “sulfonate” refers to the group SO₃H and includes groups havingthe hydrogen replaced with, for example a C₁₋₆alkyl group(“alkylsulfonate”), an aryl (“arylsulfonate”), an aralkyl(“aralkylsulfonate”) and so on. C₁₋₃sulfonates are preferred, such asfor example, SO₃Me, SO₃Et and SO₃Pr.

The term “isomers”, as used herein, refer to stereoisomers,diastereomers, enantiomers and tautomers. “Tautomers” may be isomersthat are readily interconvertable by rapid equilibrium. For example,carbonyl compounds that have a hydrogen on their alpha-carbon arerapidly interconverted with their corresponding enols.

As used herein, the terms “alkyl”, “alkenyl”, and the prefix “alk-” areinclusive of straight chain groups and branched chain groups and cyclicgroups, e.g., cycloalkyl and cycloalkenyl. Unless otherwise specified,these groups contain from 1 to 20 carbon atoms, with alkenyl groupscontaining from 2 to 20 carbon atoms. In some embodiments, these groupshave a total of at most 10 carbon atoms, at most 8 carbon atoms, at most6 carbon atoms, or at most 4 carbon atoms. Cyclic groups can bemonocyclic or polycyclic and preferably have from 3 to 10 ring carbonatoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl,cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstitutedbornyl, norbornyl, and norbornenyl.

The term “heterocyclic” includes cycloalkyl or cycloalkenyl non-aromaticrings or ring systems that contain at least one ring heteroatom (e.g.,O, S, N).

Unless otherwise specified, “alkylene” and “alkenylene” are the divalentforms of the “alkyl” and “alkenyl” groups defined above. The terms,“alkylenyl” and “alkenylenyl” are used when “alkylene” and “alkenylene”,respectively, are substituted. For example, an arylalkylenyl groupcomprises an alkylene moiety to which an aryl group is attached.

The term “haloalkyl” is inclusive of groups that are substituted by oneor more halogen atoms, including perfluorinated groups. This is alsotrue of other groups that include the prefix “halo-”. Examples ofsuitable haloalkyl groups are difluoromethyl, trifluoromethyl, and thelike. “Halogens” are elements including chlorine, bromine, fluorine, andiodine.

The term “aryl” as used herein includes monocyclic or polycyclicaromatic hydrocarbons or ring systems. Examples of aryl groups includephenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may besubstituted or unsubstituted. Aryl groups include aromatic annulenes,fused aryl groups, and heteroaryl groups. Aryl groups are also referredto herein as aryl rings.

Unless otherwise indicated, the term “heteroatom” refers to the atoms O,S, or N.

The term “heteroaryl” includes aromatic rings or ring systems thatcontain at least one ring heteroatom (e.g., O, S, N). In someembodiments, the term “heteroaryl” includes a ring or ring system thatcontains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and 0,S, and/or N as the heteroatoms. Suitable heteroaryl groups includefuryl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl,triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl,thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl,pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl,naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl,pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl,oxadiazolyl, thiadiazolyl, and so on.

The terms “arylene” and “heteroarylene” are the divalent forms of the“aryl” and “heteroaryl” groups defined above. The terms “arylenyl” and“heteroarylenyl” are used when “arylene” and “heteroarylene”,respectively, are substituted. For example, an alkylarylenyl groupcomprises an arylene moiety to which an alkyl group is attached.

The term “fused aryl ring” includes fused carbocyclic aromatic rings orring systems. Examples of fused aryl rings include benzo, naphtho,fluoreno, and indeno.

The term “annulene” refers to aryl groups that are completely conjugatedmonocyclic hydrocarbons. Examples of annulenes include cyclobutadiene,benzene, and cyclooctatetraene. Annulenes present in an aryl group willtypically have one or more hydrogen atoms substituted with other atomssuch as carbon.

When a group is present more than once in any formula or schemedescribed herein, each group (or substituent) is independently selected,whether explicitly stated or not. For example, for the formula —C(O)NR₂each of the two R groups is independently selected.

As a means of simplifying the discussion and the recitation of certainterminology used throughout this application, the terms “group” and“moiety” are used to differentiate between chemical species that allowfor substitution or that may be substituted and those that, in theparticular embodiment of the invention, do not so allow for substitutionor may not be so substituted. Thus, when the term “group” is used todescribe a chemical substituent, the described chemical materialincludes the unsubstituted group and that group with nonperoxidic O, N,S, Si, or F atoms, for example, in the chain as well as carbonyl groupsor other conventional substituents. Where the term “moiety” is used todescribe a chemical compound or substituent, only an unsubstitutedchemical material is intended to be included. For example, the phrase“alkyl group” is intended to include not only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like, but also alkyl substituents bearing furthersubstituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl,halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, “alkyl group”includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls,hydroxyalkyls, sulfoalkyls, etc. On the other hand, the phrase “alkylmoiety” is limited to the inclusion of only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like.

The invention is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Pharmaceutically acceptable” as used herein means that the compound orcomposition or carrier is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the necessity of the treatment.

The term “therapeutically effective amount” or “pharmaceuticallyappropriate dosage”, as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include oils such as com oil,buffers such as PBS, saline, polyethylene glycol, glycerin,polypropylene glycol, dimethylsulfoxide, an amide such asdimethylacetamide, a protein such as albumin, and a detergent such asTween 80, mono- and oligopolysaccharides such as glucose, lactose,cyclodextrins and starch.

The term “administering” or “administration”, as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

The term “systemic delivery”, as used herein, refers to any suitableadministration methods which may delivery the compounds in the presentinvention systemically. In one embodiment, systemic delivery may beselected from the group consisting of oral, parenteral, intranasal,inhaler, sublingual, rectal, and transdermal administrations.

A route of administration in pharmacology and toxicology is the path bywhich a drug, fluid, poison, or other substance is taken into the body.Routes of administration may be generally classified by the location atwhich the substance is applied. Common examples may include oral andintravenous administration. Routes can also be classified based on wherethe target of action is. Action may be topical (local), enteral(system-wide effect, but delivered through the gastrointestinal tract),or parenteral (systemic action, but delivered by routes other than theGI tract).

A topical administration emphasizes local effect, and substance isapplied directly where its action is desired. Sometimes, however, theterm topical may be defined as applied to a localized area of the bodyor to the surface of a body part, without necessarily involving targeteffect of the substance, making the classification rather a variant ofthe classification based on application location. In an enteraladministration, the desired effect is systemic (non-local), substance isgiven via the digestive tract. In a parenteral administration, thedesired effect is systemic, and substance is given by routes other thanthe digestive tract.

The examples for topical administrations may include epicutaneous(application onto the skin), e.g., allergy testing or typical localanesthesia, inhalational, e.g. asthma medications, enema, e.g., contrastmedia for imaging of the bowel, eye drops (onto the conjunctiva), e.g.,antibiotics for conjunctivitis, ear drops, such as antibiotics andcorticosteroids for otitis externa, and those through mucous membranesin the body.

Enteral administration may be administration that involves any part ofthe gastrointestinal tract and has systemic effects. The examples mayinclude those by mouth (orally), many drugs as tablets, capsules, ordrops, those by gastric feeding tube, duodenal feeding tube, orgastrostomy, many drugs and enteral nutrition, and those rectally,various drugs in suppository.

The examples for parenteral administrations may include intravenous(into a vein), e.g. many drugs, total parenteral nutritionintra-arterial (into an artery), e.g., vasodilator drugs in thetreatment of vasospasm and thrombolytic drugs for treatment of embolism,intraosseous infusion (into the bone marrow), intra-muscular,intracerebral (into the brain parenchyma), intracerebroventricular (intocerebral ventricular system), intrathecal (an injection into the spinalcanal), and subcutaneous (under the skin). Among them, intraosseousinfusion is, in effect, an indirect intravenous access because the bonemarrow drains directly into the venous system. Intraosseous infusion maybe occasionally used for drugs and fluids in emergency medicine andpediatrics when intravenous access is difficult.

Any route of administration may be suitable for the present invention.In one embodiment, the compound of the present invention may beadministered to the subject via intravenous injection. In anotherembodiment, the compounds of the present invention may be administeredto the subject via any other suitable sytemic deliveries, such as oral,parenteral, intranasal, sublingual, rectal, or transdermaladministrations.

In another embodiment, the compounds of the present invention may beadministered to the subject via nasal systems or mouth through, e.g.,inhalation.

In another embodiment, the compounds of the present invention may beadministered to the subject via intraperitoneal injection or IPinjection.

As used herein, the term “intraperitoneal injection” or “IP injection”refers to the injection of a substance into the peritoneum (bodycavity). IP injection is more often applied to animals than to humans.In general, IP injection may be preferred when large amounts of bloodreplacement fluids are needed, or when low blood pressure or otherproblems prevent the use of a suitable blood vessel for intravenousinjection.

In animals, IP injection is used predominantly in veterinary medicineand animal testing for the administration of systemic drugs and fluidsdue to the ease of administration compared with other parenteralmethods.

In humans, the method of IP injection is widely used to administerchemotherapy drugs to treat some cancers, in particular ovarian cancer.Although controversial, this specific use has been recommended as astandard of care.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. In addition,polymers of the present invention may also be optically active. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,“Handbook of Chemistry and Physics”, 67th Ed., 1986-87, inside cover.

As used herein, the term “subject” or “individual” refers to a human orother vertebrate animal. It is intended that the term encompass“patients.”

The term “synergistic” refers to a combination which is more effectivethan the additive effects of any two or more single agents. Asynergistic effect permits the effective treatment of a disease usinglower amounts (doses) of individual therapy. The lower doses result inlower toxicity without reduced efficacy. In addition, a synergisticeffect can result in improved efficacy. Finally, synergy may result inan improved avoidance or reduction of disease as compared to any singletherapy.

Combination therapy can allow for the product of lower doses of thefirst therapeutic or the second therapeutic agent (referred to as“apparent one-way synergy” herein), or lower doses of both therapeuticagents (referred to as “two-way synergy” herein) than would normally berequired when either drug is used alone.

As used herein, “pharmaceutically-acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic agents, absorption delaying agents, and the like.Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include oils such as com oil,buffers such as PBS, saline, polyethylene glycol, glycerin,polypropylene glycol, dimethylsulfoxide, an amide such asdimethylacetamide, a protein such as albumin, and a detergent such asTween 80, mono- and oligopolysaccharides such as glucose, lactose,cyclodextrins and starch.

The formulation used in the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. The use of such media and agents forpharmaceutically-active substances is well known in the art.Supplementary active compounds can also be incorporated into the imagingagent of the invention. The imaging agent of the invention may furtherbe administered to an individual in an appropriate diluent or adjuvant,co-administered with enzyme inhibitors or in an appropriate carrier suchas human serum albumin or liposomes. Pharmaceutically-acceptablediluents include sterile saline and other aqueous buffer solutions.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin inhibitor,diethylpyrocarbonate, and trasylol. Liposomes inhibitors includewater-in-oil-in-water CGF emulsions, as well as conventional liposomes(see J. Neuroimmunol. 1984, 7, 27).

As described herein, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,naphthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. See J. Pharm. Sci. 1977, 66, 1-19.

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, J. Pham. Sci. 1977., supra)

More specifically, the compounds that can be formulated into apharmaceutical composition include a therapeutically-effective amount ofthe first compound, a therapeutically effective amount of the secondcompound, and a pharmaceutically-acceptable carrier. Thetherapeutically-effective amount of the compounds and the specificpharmaceutically-acceptable carrier will vary depending upon, e.g., theage, weight, sex of the subject, the mode of administration, and thetype of viral condition being treated.

In a particular aspect, the pharmaceutical composition which can be usedincludes the compounds of the present invention in effective unit dosageform. As used herein, the term “effective unit dosage” or “effectiveunit dose” is used herein to mean a predetermined amount sufficient tobe effective against AD or the like. Examples include amounts thatenable treatment of amyloid deposit(s) in vivo or in vitro that yieldacceptable toxicity and bioavailability levels for pharmaceutical use,and/or prevent cell degeneration and toxicity associated with fibrilformation.

The pharmaceutical compositions may contain the first compound or thesecond compound used in the method of this invention in an amount offrom 0.01 to 99% by weight of the total composition, preferably 0.1 to80% by weight of the total composition. For oral administration, thefirst compound or the second compound is generally administered in anamount of 0.1 g/body to 15 g/body, preferably 0.5 g/body to 5 g/body.For intravenous injection, the dose may be about 0.1 to about 30mg/kg/day, preferably about 0.5 to about 10 mg/kg/day. If appliedtopically as a liquid, ointment, or cream, the first compound or thesecond compound may be present in an amount of about 0.1 to about 50mg/mL, preferably about 0.5 to 30 mg/mL of the composition.

For systemic administration, the daily dosage as employed for adulthuman treatment will range from about 0.1 mg/kg to about 150 mg/kg,preferably about 0.2 mg/kg to about 80 mg/kg.

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

EXAMPLES

It should be understood that the above-described embodiments and thefollowing examples are given by way of illustration, not limitation.Various changes and modifications within the scope of the presentinvention will become apparent to those skilled in the art from thepresent description.

Example 1 Cromolyn and Ibuprofen Combination Treatment

The following is a dosing example calculation for cromolyn:

-   -   1. Effective comolyn administration for AD treatment is        significantly different from that of cromolyn for lung        inflammation and asthma. For lung inflammation or asthma        subjects use inhalation devices 1-4 times per day; each inhaled        dose contains 20 mg of dry powder.    -   2. The dry cromolyn powder (usual size is >5 microns) is        formulated with lactose with much larger size.    -   3. When inhaled, dry cromolyn powder is separated from the        lactose via the inhaler device action (spinhaler, cyclohaler, or        monodose inhalers) or in the upper part of the airway and the        dry cromolyn is delivered to the lung.    -   4. Cromolyn has also been delivered as a solution that enters        via the gastric system.

For AD, these treatments will not result in any significant action on ADprogression or modification.

For AD treatment, to be effective, the drug has to be deliveredsystemically to allow for brain uptake. Therefore, cromolyn dry powderhas to be <3 microns. In certain embodiments, cromolyn is from about 0.5microns to about 1.5 microns in diameter. Powders of this size willreach the alvular nodes and be delivered systemically.

The estimated dose for daily treatment is about 16 mg per subject perday, about 4 mg per subject per day, or about 1 mg per subject per day.

From a preliminary biodistribution of a cromolyn analog labeled withF-18, the brain uptake is about 1% dose per gram in the brain.Therefore, it is estimated that from a 16 mg dose, for 1 mg takensystematically, 0.01 mg will be taken in the brain. The estimated doseper gram of brain will be 0.01/1500 gram (average brain mass); thisequals 7 ng/gram of brain. This amount slows down polymerization of thedaily Aβ peptide produced in the brain. Therefore, amounts that are 5times lower than the 80 mg used for asthma treatment, should beeffective. Cromolyn has a LogP of 1.9, PSA of 189, and % PSA of 44(JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 6, JUNE 2003).

Inhibition data showed that cromolyn inhibits Aβ peptide polymerizationby 8 fold. Other inhibition experiments showed that cromolyn inhibits Aβpeptide polymerization in 5 nM concentrations.

In combination with cromolyn, a dose of 2 mg of ibuprofen could be givenas pill, capsule, or liquid. This low dose of ibuprofen is sufficient totreat the invisible inflammation response to the Aβ peptide. Higherdoses may work initially but may worsen the AD subjects in the long run.

Example 2 In Vivo Experiments of Cromolyn and Ibuprofen CombinationTreatment

Three mice groups (five animals in each) were tested in a Morris waternavigation test. Two groups were four months young APP/PS1 including amutant Aβ mouse and a model indicative of Alzheimer's Diseaseprogression. One APP/PS1 group was treated with Cromolyn and ibuprofencombination for six months, and the second was untreated as an controlgroup and a third untreated wild type was used as a normal control. FIG.1 is a graph showing the in-vivo study summary. WT (wild type, rightpanel) shows normal untreated mice. The control group (left panel) showstransgenic mice that did not received drug treatment. The treated group(Mid panel) shows transgenic mice that receivedAZLT-OP1(cromolyn+ibuprofen) for six month by Intraperitoneal (IP)injection twice weekly. Mice were trained for 7 days to remember thelocation of the platform. At day 8, the platform was removed, and thetimes of crossing the platform area was recorded.

In another study, 7.5 month old APP/PS1 mice completed treated for aweek as an acute treatment using three different doses of CromolynSodium (1.05 mg/kg, 2.1 mg/kg and 3.15 mg/kg). The treatment was givenby IP injection everyday for 7 days before sacrificing the mice andharvesting the brain. Brain extracts were quantified for the totalamount of Aβ40, Aβ42 and Aβ oligomers. FIG. 4 depicts the results of invivo cromolyn and ibuprofen treatment of transgenic mice modeling likeAlzheimer'Disease.

Here are the main conclusions of this acute study:

-   -   1. A dose-dependent decrease in the amount of Aβ40 and Aβ42        associated with the two higher doses (2.1 mg/kg and 3.15 mg/kg),        up to 50% was observed.    -   2 This effect was sustained after treatment of the samples with        guanidine-HC1 to dissolve any amyloid aggregates.    -   3 The quantification of oligomeric species using the 82E1/82E1        ELISA kit failed to show any difference among the experimental        groups.

One explanation to the insignificant change is that acute exposure toCromolyn Sodium treatment primarily affects monomeric species, impactingoligomers or higher-order aggregates chronic longer treatment term.Acute treatment would not cause a substantial change in the oligomericquantities.

Example 3 Cromolyn Derivatives for Inhibiting Polymerization ofAlzheimer's Disease Oligomers and Treating Alzheimer's Disease

In another experiment, cromolyn derivatives were tested as inhibitors ofAβ polymerization. Inhibiting Aβ oligomer production will provide ofAlzheimer's Disease and treating Alzheimer's Disease.

The investigational product ALZT-OP1a (cromolyn sodium) is a syntheticchromone derivative that has been approved for use by the FDA since the1970s for the treatment of asthma. For asthma treatment, cromolyn sodiumpowder was micronized for inhalation to the lungs via dry powderinhaler, i.e. the Spinhaler device. Liquid intranasal and ophthalmicformulations have also been developed for the treatment of rhinitis andconjunctivitis.

The mechanism of action for cromolyn sodium (ALZT-OP1a) is characterizedas a mast cell stabilizer, namely to suppress cytokine release fromactivated lymphocytes together with preventing the release of histaminefrom mast cells (Netzer, 2012; Keller, 2011). It was administered fourtimes daily as prophylaxis for allergic and exercise-induced asthma, notas a treatment for acute attacks.

Applicants have discovered a new mechanism of action for cromolyn,which, along with its role for suppressing immune responses, enables there-purposing of this approved drug for use to halt AD progression. TheApplicants' studies have shown that cromolyn sodium binds tobeta-amyloid peptides and inhibits its polymerization into oligomers andhigher order aggregates. The inhibition of beta-amyloid polymerizationwill arrest amyloid-mediated intoxication of neurons and restore thepassage of these aberrant beta-amyloid oligomers out of the brain ratherthan their accumulation.

Applicants' studies showed that cromolyn or its derivatives penetratesthe blood-brain barrier in animal models, so that plasma bioavailabilityfollowing cromolyn inhalation will translate to concentrations in thebrain sufficient to interfere with beta-amyloid oligomerization andaccumulation. Inhalation of cromolyn sodium was shown to be the mosteffective non-injected administration route for systemic bioavailabilityof cromolyn sodium in animals and humans (Moss, 1970; Neale, 1986;Richards, 1987; Aswania, 1999; Tronde,2003). An FDA-approved route ofadministration for cromolyn sodium is oral inhalation using acapsule-based dry powder inhaler, with 20 mg cromolyn sodium loaded percapsule. Studies have shown that with high inspiratory rates, theinhaled cromolyn sodium is delivered efficiently to the human lung, with10-15% of the inhaled drug-delivered-dose absorbed into the bloodstream(Richards, 1987; Keller, 2011). For these reasons, cromolyn sodiuminhalation with a dry powder inhaler device was selected as the route ofadministration in the present invention. However, plasma levels ofcromolyn following inhalation are reported to show high intra- andinter-subject variability, and that cromolyn uptake by asthmatics waslower than in healthy volunteers (Richards, 1987; Keller, 2011).

For planned human studies, each blister will contain the active productingredient (cromolyn sodium) and inhalation grade lactose monohydrate asan excipient. The once-daily cromolyn dose to be tested in this study isless than 20% the dose from the four-times daily approved dose level (80mg cromolyn sodium total per day) for the treatment of asthma.

Taken together, the once daily ALZT-OP1a dose in this study shouldpreserve the drug's excellent safety and tolerability profile, yet ispredicted to achieve the nanomolar drug concentrations needed to blockbeta-amyloid oligomerization in the brain to prevent Alzheimer's diseaseprogression.

Example 4 Cromolyn Derivatives for Inhibiting Polymerization ofAlzheimer's Disease Oligomers

FIG. 5 illustrates the measurement of TBS soluble Aβ level by WAKOELISA. The experiments show that TBS Aβ level decreases following bytreatment of cromolyn sodium with dose-dependency. FIG. 5A shows thatAβ40 level decreases following by treatment of cromolyn sodium withdose-dependency. FIG. 5B shows that Aβ42 level decreases following bytreatment of cromolyn sodium with dose-dependency. As indicated, thenumber of animals per group is N=3 or 5, average±SE. The p value issignificant using one-way ANOVA test (Bonferroni's test). Both of totalsoluble Aβ [as shown as Gdn+(Guanidine-HC1)] and monomeric Aβ [as shownas Gdn-(no guanidine)] decease after the addition of cromolyn sodium.The dose of 2.1 mg/kg of cromolyn sodium was enough to decrease TBSsoluble Aβ.

FIG. 6 illustrates the measurement of TBS soluble Aβ oligomer level IBLoligomer ELISA (82E1-82E1). The experiments show that Aβ oligomer levelwas not changed following the treatment of cromolyn sodium. FIG. 6Ashows the experiments of IBL Aβ oligomer ELISA (82E1-82E1). FIGS. 6B and6C show the difference the experiments with Gdn and those without Gdnusing Aβ WAKO ELISA. N=3 or 5 animals per group, average±SE. The p valueis not significant using one-way ANOVA test (Bonferroni's test). BothELISA (IBL oligomer ELISA and the differences between with and withoutGdn using WAKO ELISA) showed that oligomer level was not changedfollowing the treatment of cromolyn sodium.

Example 5 Discussion

Applicants summarize the rationale behind the treatment as follows:

1. Molecular structure is similar to some that had affinity to plaque(Foruma I and table 1). The significant difference is that the drug inthe present invention works in nanomolar concentrations as compared tomicromolar concentrations of other previous drugs.

TABLE 1 The structural similarity of fisetin analogues and their effecton Aβ fibril formation. Substituents Effects on Aβ Compound 3 5 7 3’ 4’5’ fibril formation Fisetin OH H OH OH OH H Inhibitory3’,4’,7-Trihydroxyflavone H H OH OH OH H Inhibitory3,3’,4’-Trihydroxyflavone OH H H OH OH H Inhibitory3,3’,7-Trihydroxyflavone OH H OH OH H H Enhancing s-Deoxykaempferol OH HOH H OH H Enhancing Luteolin H OH OH OH OH H Inhibitory Quercetin OH OHOH OH OH H Inhibitory Chrysin H OH OH H H H Enhancing Kaempferol OH OHOH H OH H Enhancing Myricetin OH OH OH OH OH OH Inhibitory

2. The suitable molecular weight of the molecules in the presentinvention allows the molecules to penetrate brain.

Chemical Structure:

Molecular Formula: C₂₃H₁₄Na₂O₁₁Molecular Weight: 512.34 [g/mol]

3. The molecules in the present invention have desirable lipophilicity(LogP) and pressure surface area (PSA) in the brain penetration range(Table 2). The PIB analog TS3124 has a 4% brain concentration, and ahigher LogP value in a range that there is no usal uptake. This isbalanced by the much lower PSA. Log P was determined by Chemdraw pro,Version 10. PSA was determined by the previous methods(http://www.daylight.com/meetings/emug00/Ert1/tpsa.html).

TABLE 2 The moelcular structures, molecular weight, lipophilicity (LogP)and pressure surface area (PSA). compound Structure Mw logP PSA PKaTS734

466.41 2.1 127.20 C0399

508.38 1.39 for diacid 125.43 TS3124

302.37 3.92  45.15 OH: 9.2 NH: 19.2

4. Mice biodistribution of radiolabeled cromolyn biodistribution shows1% dose per gram brain accumulate. FIG. 7 illustrates thebiodistribution of radiolabeled cromolyn Compound A followingintravenous injection in mice. In FIG. 7, a 5, 30 or 60 minute,corresponding to Series 1, 2 or 3, respectively in the graph, brainuptake shows 1% accumulation with little or no washout for the periodmeasured.

5. The binding of cromolyn to Aβ and its polymerization inhibition wasconfirmed by four independent methods.

UV Aggregation Assay

Abeta peptide aggregation and the impact of drugs to slow or preventAbeta aggregation was measured by a UV absorbance assay (Findeis, 1999).Abeta (1-40) peptides, at 50 μM, were mixed with 50 μM drug in assaybuffer and the plate was incubated at ambient temperature on a platereader. The UV absorbance was monitored at 540 nm over a 2-3 h period.

Polymerization of Aβ-monomer peptides into clusters of trimers andtetramers initiates the Aβ aggregation process into protofibrils andthen into fibrils that form amyloid plaques. The polymerizationexperiments revealed that Aβ monomer reached 50% polymerization in 14minutes. At equimolar concentrations with Aβ, the addition of cromolyninhibited the rate of AP polymerization 7-fold, namely 50%polymerization required 75 minutes incubation, compared to 14 minutes inthe absence of drug.

TABLE 3 Cromolyn inhibits Aβ polymerization. Relative Increase in %Thioflavin T Relative Polymerization Test Compound Bound Binding Time(fold) Vehicle 37% 1   1   TS734 (cromlyn) 30% 0.82 7.8

LC/MS/MS Binding Assay

Binding was measured by equilibrium dialysis. Amyloid fibrils werepreformed by incubating the peptide in buffer with shaking for 120 h at27° C. The drugs were incubated with fibrils (50 μM peptide) in a REDequilibrium dialysis device (Pierce), and the amount of test agent oneach side is determined by LC/MS/MS. Percent bound was calculated as1−(free conc/total conc) after correcting for background signal.Thioflavin-T was used as a positive control. Binding is displacement ofThioflavin T. Polymerization is ranked for relative Aβ. In general,compounds that rank highly in inhibiting polymerization rank low inbinding to aggregates, and vice versa.

Competition Binding Assay

The competition assay was performed as described previously (Ono andHayashi, 2009). Amyloid peptide aggregates were preformed by incubatingAβ (1-40) peptide with buffer for 3 days at 37° C. Drugs at 20 μM weremixed with assay solution containing 10 μg/mL amyloid peptide aggregates+3 μM Thioflavin-T on one side of a RED dialysis device with assaybuffer added to the other side. After 4h dialysis, the amount ofThioflavin-T was determined by LC/MS/MS. The relative binding wasdetermined by normalizing the percent binding by the percent binding ofthe vehicle control.

Aβ Aggregation by Thioflavin T Assay

One of the most routinely used approaches to monitor Aβ polymerizationis the thioflavin T binding assay. When thioflavin T binds to beta-sheetrich structures, such as amyloid aggregates, the dye displays enhancedfluorescence and a characteristic red shift in its emission spectrum. Aβpeptide at 5 μM was mixed with 10 μM thioflavin T with drug at differentconcentrations. In the absence of drug, Aβ polymerization showsincreasing thioflavin T fluorescence over 60-180 min, as shown in FIG.5.

The addition of cromolyn (C0399) and its ¹⁸F derivative (TS734) atnanomolar concentration shows inhibition of Aβ aggregation, as shown inFIG. 6.

By four separate in vitro assays, cromolyn sodium, at nanomolarconcentrations, effectively inhibits Aβ amyloid peptide polymerizationinto oligomers and higher order aggregates.

6. Preliminary analysis of the binding model indicates that cromolynbinding to the surface of beta sheet across the beta strand in a mannersimilar to Thioflavin-T. FIGS. 10 and 11 illustrate the side and topview of the relative structures and locations of cromolyn and Aβ aftercromolyn binds Aβ through a binding model simulation.

7. Applicants tested several other structures for treating AD inaddition to cromolyn. Several types of compounds for both imaging andtherapeutic agents have been evaluated for Aβ peptide polymerizationinhibition.

In an effort to combine bioavailability and dual function, Applicantshave tethered scyllo-inositol, which is transported across theblood-brain barrier and known to bind and neutralize oligomers intosoluble complexes (McLaurin, Kierstead, et al., 2006; Sun, Zhang, etal., 2008), to 2-ethyl-8-methyl-2,8-diazospiro-4,5-decan-1,3-dione, amuscarinic M1 receptor agonist (Palacios, Bolliger, et al., 1986). RS-86was chosen because evidence has shown that it improves cognitivefunction, mood and social behavior in some AD patients (Wettstein andSpiegel, 1985). M2 receptors function in cholinergic nerve terminals toregulate the release of acetylcholine, whereas M1 receptors are locatedon postsynaptic cells and facilitate cellular excitation (Mash, Flynn,1985). Since presynaptic cholinergic neurons degenerate in AD whilepostsynaptic M1 muscarinic receptors remain in tact, the use oflong-acting muscarinic agonists like RS-86 has been proposed as atreatment strategy for memory loss. However, RS86 has low brainpenetration; combining it with inositol using a linkage which can bemetabolized once in the brain may increase bioavailability of theagonist as well as maintaining the beneficial effect of inositol. In thepast, both inositol, in the form of 1-fluoro-scyllo-inositol, and RS-86derivatives have been radiolabeled with F-18 or C-11 as potential PETprobes for AD.

8. It is believed that these suitable compounds target mast cells byinhibiting cytokine production therefore an additional treatment theinflammatory response associated with the AD trigger and process. Intheir previous publication (Jin, Silverman, et al. 2009), Jin andco-workers indicate that the potential cromolyn compounds can be used asa Mast cell inhibitors.

Example 6 Non-Steroidal Anti-Inflammatory drugs (NSAIDs)

Compelling evidence from multiple epidemiology studies revealed thatlong-term dosing with non-steroidal anti-inflammatory drugs (NSAIDs)dramatically reduced AD risk in the elderly, including delayed diseaseonset, reduced symptomatic severity and slowed cognitive decline (Veld,2001; Etminan, 2003; Imbimbo, 2010). Three mechanisms have been proposedhow NSAIDs inhibit the processes that contribute to AD progression: i)by inhibiting COX activity to reduce or prevent microglial activationand cytokine production in the brain (Mackenzie, 1998; Alafuzoff, 2000;Yan, 2003; Gasparini, 2004; Imbimbo, 2010); ii) by reducing amyloiddeposition (Weggen, 2001; Yan, 2003; Imbimbo, 2010); or iii) by blockingCOX-mediated prostaglandin E2 responses in synapses (Kotilinek, 2008).

Therefore, NSAIDs are predicted to dampen the neuro-inflammatoryresponse and impact AD progression via several mechanisms. Whenadministered together with drugs that inhibitbeta-amyloidoligomerization, the combination treatment paradigm isproposed to attenuate the multiple triggers leading to neurodegenerationand neuronal death. The decline in cognitive performance may bereversed, due to neuronal plasticity and neurogenesis in the hippocampus(Kohman, 2013), if AD progression is arrested at a very early stage.

Ibuprofen

Ibuprofen is a non-selective COX inhibitor for treating inflammation asa non-steroidal anti-inflammatory drug (NSAID). The COX enzymes convertcertain fatty acids to prostaglandins. The prostaglandins at the end ofthe “chain” of reactions that starts with the COX enzyme cause anincreased sensitivity to pain, fever, and vasodilation (increased bloodflow or inflammation). By inhibiting the start of this chain ofreactions, ibuprofen therefore reduces pain, fever, and inflammation.Because ibuprofen blocks the activity of both COX enzymes, it isconsidered a non-selective COX inhibitor NSAID.

ALZT-OP1 therapy for the treatment of individuals with amnestic mildcognitive impairment. ALZT-OP1 is a multi-functional drug therapyconsisting of cromolyn sodium (ALZT-OP1a) administered by inhalation toinhibit beta-amyloid peptide polymerization and to dampen immuneresponses, plus a concomitant but separately administered low dose oralibuprofen tablet (ALZT-OP1b) to inhibit the neuro-inflammatory responsein persons with confirmed amnestic mild cognitive impairment (aMCI) dueto Alzheimer's disease. Both active pharmaceutical ingredient (API)drugs in this ALZT-OP1 formulation are approved, marketed drugs thathave been re-purposed for use to prevent the onset of dementia andAlzheimer's disease progression.

ALZT-OP1a

The investigational product ALZT-OP1a (cromolyn sodium) is a syntheticchromone derivative that has been approved for use by the FDA since the1970s for the treatment of asthma. For asthma treatment, cromolyn sodiumpowder was micronized for inhalation to the lungs via dry powderinhaler, i.e., the Spinhaler device. Liquid intranasal and ophthalmicformulations have also been developed for the treatment of rhinitis andconjunctivitis.

The mechanism of action for cromolyn sodium (ALZT-OP1a) is characterizedas a mast cell stabilizer, namely to suppress cytokine release fromactivated lymphocytes together with preventing the release of histaminefrom mast cells (Netzer, 2012; Keller, 2011). It was administered fourtimes daily as prophylaxis for allergic and exercise-induced asthma, notas a treatment for acute attacks.

We have discovered a new mechanism of action for cromolyn, which, alongwith its role for suppressing immune responses, enables the re-purposingof this approved drug for use to halt AD progression. Our studies haveshown that cromolyn sodium binds to beta-amyloid peptides and inhibitsits polymerization into oligomers and higher order aggregates. Theinhibition of beta-amyloid polymerization will arrest amyloid-mediatedintoxication of neurons and restore the passage of these aberrantbeta-amyloid oligomers out of the brain rather than their accumulation.

Our studies showed that cromolyn penetrates the blood-brain barrier inanimal models, so that plasma bioavailability following cromolyninhalation will translate to concentrations in the brain sufficient tointerfere with beta-amyloid oligomerization and accumulation. Inhalationof cromolyn sodium was shown to be the most effective non-injectedadministration route for systemic bioavailability of cromolyn sodium inanimals and humans (Moss, 1970; Neale, 1986; Richards, 1987; Aswania,1999; Tronde,2003). An FDA-approved route of administration for cromolynsodium is oral inhalation using a capsule-based dry powder inhaler, with20 mg cromolyn sodium loaded per capsule. Studies have shown that withhigh inspiratory rates, the inhaled cromolyn sodium is deliveredefficiently to the human lung, with 10-15% of the inhaleddrug-delivered-dose absorbed into the bloodstream (Richards, 1987;Keller, 2011). For these reasons, cromolyn sodium inhalation with a drypowder inhaler device was selected as the route of administration inthis study. However, plasma levels of cromolyn following inhalation arereported to show high intra- and inter-subject variability, and thatcromolyn uptake by asthmatics was lower than in healthy volunteers(Richards, 1987; Keller, 2011).

Cromolyn sodium powder blend (ALZT-OP1a) will be loaded into blistersfor use with a dry powder inhaler with reproducible aerosol performanceat a range of inspiratory rates. Each blister will contain the activeproduct ingredient (cromolyn sodium) and inhalation grade lactosemonohydrate as an excipient. The once-daily cromolyn dose to be testedin this study is less than 20% the dose from the four-times dailyapproved dose level (80 mg cromolyn sodium total per day) for thetreatment of asthma. The dose is calculated to titrate the estimateddaily 22-27 nanogram of Aβ amyloid plaque produced in the brain.

Taken together, the once daily ALZT-OP1 a dose in this study shouldpreserve the drug's excellent safety and tolerability profile, yet ispredicted to achieve the nanomolar drug concentrations needed to blockbeta-amyloid oligomerization in the brain to prevent Alzheimer's diseaseprogression.

ALZT-OP1b (ibuprofen). The generic name is iso-butyl-propanoic-phenolicacid. ALZT-OP1b is an over the counter drug, taken in orally and doesnot require prescription. Ibuprofen has a long safety history. The drugis used for pain, fever, sports injuries and gastrointestinal problems.The weight dosage independence has been indicated on the drug package.

The investigational product ALZT-OP1b (ibuprofen) is non-selective COXinhibitor for treating inflammation as a non-steroidal anti-inflammatorydrug (NSAID). The COX enzymes convert certain fatty acids toprostaglandins. The prostaglandins at the end of the “chain” ofreactions that starts with the COX enzyme cause an increased sensitivityto pain, fever, and vasodilation (increased blood flow or inflammation).By inhibiting the start of this chain of reactions, ibuprofen thereforereduces pain, fever, and inflammation. Because ibuprofen blocks theactivity of both COX enzymes, it is considered a non-selective COXinhibitor NSAID.

As described above, dampening the neuro-inflammatory response willimpact AD progression by several mechanisms. Ibuprofen, which crossesthe human blood brain barrier (Bannworth, 1995; Parepally, 2006),dampens the production of pro-inflammatory cytokines (Gasparini, 2004),which should contribute to its utility for preventing AD progression.However, NSAIDs, such as rofecoxib and naproxen, for the treatment of ADhas been inconclusive or contributed to higher risk of AD progressionwhen administered as the sole therapy in clinical trials (Thal, 2005;Imbimbo, 2010) despite the multiple epidemiology studies showing reducedAD risk in individuals taking NSAIDs, including ibuprofen (Veld, 2001;Etminan, 2003). Besides the criticism surrounding the choice ofrofecoxib and naproxen as the NSAIDs for sole therapy in AD (Gasparini,2004), the ADAPT rofecoxib/naproxen treatment trial was conducted withsubjects exhibiting mild-to-moderate AD (Aisen 2003; Breitner, 2011).Given the epidemiology data, it has been hypothesized that NSAIDadministration may be beneficial only very early in disease (Imbimbo,2010; Breitner, 2011). The aMCI patient population is therefore thegroup that we have selected to be tested in this clinical study.

It is important to note that in the NSAID epidemiology studies, AD riskdecrease was restricted to NSAIDs that presumably lower beta-amyloid(42-)peptide levels, such as ibuprofen and indomethacin (Gasparini,2004; Imbimbo, 2010), and long-term dosing with low NSAID doses wereequally effective as higher doses (Broe, 2000; Breitner 2001). Hence, inone cohort in this AZTherapies ALZT-OP1 trial, oral ibuprofen will beadministered as tablets (ALZT-OP1b) at a dose lower (less than 5%) ofthe lowest over-the-counter approved dose. In combination with cromolynsodium inhalation treatment (ALZT-OP1a), we will test the hypothesisthat dampening the low level neuroinflammatory response with ibuprofenwill contribute significantly to preventing cognitive decline due toAlzheimer's disease progression. The dose is calculated to titrate theestimated invisible inflammatory response at the early stages of thedisease.

Uncontrolled ibuprofen dosage is associated with several side effectssuch as nausea, headache, ulcers, dizziness, and hypertension. A minornumber of cases can cause heart or renal failures. The overdose ofibuprofen can be dangerous. The proposed daily dose for this clinicaltrial is 20 fold lower than the dose over the counter, and the totalyearly dose totaled from the chronic daily dose is less than a totalweekly dose over the counter. It is not expected that the yearlytoxicity will exceed the weekly over the counter dose.

Risk benefits of ALZT-OP1 (cromolyn)

The main goal for using ALZT-OP1 in aMCI subject is its predictedmultifunctional treatment of the early appearance signs of cognitiveimpairment associated with Alzheime's Disease. Low dose of ALZT-OP1a isexpected to control Aβ oligomerization and slow down the extra cellularAβ fibril brain accumulation. At the same time, low dose of ALZT-OP1acan inhibit cytokine production from the high brain must cellconcentration. The low dose ALZT-OP1b (ibuprofen), a known non-specificCOX inhibitor, is expected to control the inflammatory responseassociated with Aβ plaque formation. The main benefits of the low dosechronic daily use are to control and slow down the earlier ADpathophysiology cascade of the main events that trigger intracellulartau tangles and neuron degeneration. ALZT-OP1 treatment will slow downlater AD stages manifestation, prolong the patient's life, bettercontrol the quality of life and significantly lower the expensive costof family and nursing treatment and human resources.

Both medications are approved for treatment since the seventies. Bothdrugs displayed excellent safety profile at much higher dosages.However, each of the drugs have its own short and chronic treatment sideeffects for the used dosages.

AZLT-OP1a has a long history of safety in adults and children. Cromolynsodium is available as metered-dose inhalers, and used for long-termasthma prevention ad control by decreasing inflammation and improvinglung function. Cromolyn blocks cytokine release of mast cells that causeairways inflammation. The drug is associated with very mild sideeffects, like coughing, skin rash, and headaches. The treatment doses inthis clinical trial are 4-8 folds lower that prescribed and are notexpected to cause any significant higher toxicity that the asthma dose.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

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1.-20. (canceled)
 21. A method of inhibiting the formation of amyloidoligomers and/or amyloid plaques associated with Alzheimer's disease ina subject in need thereof comprising administering to the subject aneffective amount of cromolyn or a salt thereof.
 22. The method of claim21, wherein the amyloid oligomers are amyloid-β oligomers.
 23. Themethod of claim 21, wherein the amyloid plaques are amyloid-β plaques.24. The method of claim 21, wherein the subject is suffering from theearly stages of Alzheimer's disease.
 25. The method of claim 21, whereinthe subject is suffering from Alzheimer's disease.
 26. The method ofclaim 21, wherein the subject is at risk of developing Alzheimer'sdisease.
 27. The method of claim 21, wherein the cromolyn or saltthereof is cromolyn sodium.
 28. The method of claim 21, wherein thecromolyn or salt thereof is formulated as a dry powder for inhalation.29. The method of claim 28, wherein the cromolyn or salt thereof isformulated for inhalation as a dry powder of less than 3 microns inparticle size.
 30. The method of claim 21, wherein the cromolyn or saltthereof is administered in an amount of about 16 mg/day or about 20mg/day.
 31. A method of inhibiting the formation of amyloid oligomersand/or amyloid plaques associated with Alzheimer's disease in a subjectin need thereof comprising administering to the subject an effectiveamount of a compound having the structure:

or a salt thereof.
 32. The method of claim 31, wherein the amyloidoligomers are amyloid-β oligomers.
 33. The method of claim 31, whereinthe amyloid plaques are amyloid-β plaques.
 34. The method of claim 31,wherein the subject is suffering from the early stages of Alzheimer'sdisease.
 35. The method of claim 31, wherein the subject is sufferingfrom Alzheimer's disease.
 36. The method of claim 31, wherein thesubject is at risk of developing Alzheimer's disease.
 37. The method ofclaim 31, wherein the compound or salt thereof is a sodium salt of thecompound.
 38. The method of claim 31, wherein the compound or saltthereof is formulated as a dry powder for inhalation.
 39. The method ofclaim 38, wherein the compound or salt thereof is formulated forinhalation as a dry powder of less than 3 microns in particle size. 40.The method of claim 31, wherein the compound or salt thereof isadministered in an amount of about 16 mg/day or about 20 mg/day.